Optogenetics (reviewed by Deisseroth. Nat Methods 8 (1): 26-9, 2011), refers to a rapidly adapted approach of using new high-speed optical methods for probing and controlling genetically targeted neurons within intact neural circuits. Optogenetics involves the introduction of light-activated channels and enzymes that allow manipulation of neural activity with millisecond precision while maintaining cell-type resolution through the use of specific targeting mechanisms. Because the brain is a high-speed system, millisecond-scale temporal precision is central to the concept of optogenetics, which allows probing the causal role of specific action potential patterns in defined cells.
As traditional genetics has made use of “loss-of-function” or “gain of function” changes that result to determine the role and expression pattern of a particular protein. Similarly, optogenetics by definition will allow addition or deletion of precise activity patterns within specific cells in the brains of intact animals, including mammals in order to probe the role of a particular neural function. By achieving photonic control of neuronal firing control the action potential patterns involved in mammalian behavior can be determined and manipulated.
Light control of motility behavior (phototaxis and photophobic responses) in green flagellate algae is mediated by sensory rhodopsins homologous to phototaxis receptors and light-driven ion transporters in prokaryotic organisms. In the phototaxis process, excitation of the algal sensory rhodopsins leads to generation of transmembrane photoreceptor currents. When expressed in animal cells, the algal phototaxis receptors function as light-gated cation channels, which has earned them the name “channelrhodopsins”. Channelrhodopsins have become useful molecular tools for light control of cellular activity.
Originally, the source of these light-activated channels and enzymes were several microbial opsins, including, Channelrhodopsin-2 (ChR2) a single-component light-activated cation channel from algae, which allowed millisecond-scale temporal control in mammals, required only one gene to be expressed in order to work, and responded to visible-spectrum light with a chromophore (retinal) that was already present and supplied to ChR2 by the mammalian brain tissue. The experimental utility of ChR2 was quickly proven in a variety of animal models ranging from behaving mammals to classical model organisms such as flies, worms, and zebrafish, and hundreds of groups have employed ChR2 and related microbial proteins to study neural circuits.
Four channelrhodopsins have been identified to date, ChR1 and ChR2 from Chlamydomonas reinhardtii (Sineshchekov, O. A., K.-H. Jung, and J. L. Spudich. Two rhodopsins mediate phototaxis to low- and high-intensity light in Chlamydomonas reinhardtii. Proc. Natl. Acad. Sci. USA. 99:8689-869, 2002.; Nagel, G., D. Ollig, M. Fuhrmann, S. Kateriya, A. M. Musti, E. Bamberg, and P. Hegemann. Channelrhodopsin-1: a light-gated proton channel in green algae. Science. 296:2395-2398, 2002; Nagel, G., T. Szellas, W. Huhn, S. Kateriya, N. Adeishvili, P. Berthold, D. Ollig, P. Hegemann, and E. Bamberg. Channelrhodopsin-2, a directly light-gated cation-selective membrane channel. Proc. Natl. Acad. Sci. USA. 100:13940-13945, 2003; Suzuki, T., K. Yamasaki, S. Fujita, K. Oda, M. Iseki, K. Yoshida, M. Watanabe, H. Daiyasu, H. Toh, E. Asamizu, S. Tabata, K. Miura, H. Fukuzawa, S. Nakamura, and T. Takahashi. Archaeal-type rhodopsins in Chlamydomonas: model structure and intracellular localization. Biochem. Biophys. Res. Commun. 301:711-717, 2003), and VChR1 and VChR2 from Volvox carteri (Zhang, F., M. Prigge, F. Beyriere, S. P. Tsunoda, J. Mattis, O. Yizhar, P. Hegemann, and K. Deisseroth. Red-shifted optogenetic excitation: a tool for fast neural control derived from Volvox carteri. Nat. Neurosci. 11:631-633, 2008; Kianianmomeni, A., K. Stehfest, G. Nematollahi, P. Hegemann, and A. Hallmann. Channelrhodopsins of Volvox carteri are photochromic proteins that are specifically expressed in somatic cells under control of light, temperature, and the sex inducer. Plant. Physiol. 151:347-366, 2009). They contain a 7-transmembrane-helix (7TM) domain characteristic of type 1 rhodopsins (Spudich, J. L., C.-S. Yang, K.-H. Jung, and E. N. Spudich. Retinylidene proteins: structures and functions from archaea to humans. Annu. Rev. Cell Dev. Biol. 16:365-392, 2000) followed by a conserved but more variable extended C-terminal region. The property of light-gated ion permeability exhibited by their 7TM domains, makes channelrhodopsins valuable tools for light-induced depolarization of cell membranes. When transfected into and expressed in excitable cells, e.g. defined subpopulations of rodent brain neurons, channelrhodopsins enable targeted light-activation of neuron firing in tissue culture and in living organisms (Boyden, E. S., F. Zhang, E. Bamberg, G. Nagel, and K. Deisseroth. Millisecond-time scale, genetically targeted optical control of neural activity. Nat. Neurosci. 8:1263-1268, 2005; Li, X., D. V. Gutierrez, M. G. Hanson, J. Han, M. D. Mark, H. Chiel, P. Hegemann, L. T. Landmesser, and S. Herlitze. Fast noninvasive activation and inhibition of neural and network activity by vertebrate rhodopsin and green algae channelrhodopsin. Proc. Natl. Acad. Sci. USA. 102:17816-17821, 2005; Nagel, G., M. Brauner, J. F. Liewald, N. Adeishvili, E. Bamberg, and A. Gottschalk. Light activation of channelrhodopsin-2 in excitable cells of Caenorhabditis elegans triggers rapid behavioral responses. Curr. Biol. 15:2279-2284, 2005). This optogenetic approach, offers temporal and spatial resolution superior to that of conventional electrical or chemical stimulation. Today, channelrhodopsins are widely used in both neuronal and non-neuronal systems, such as glial, muscle and embryonic stem cells as a tool for controlled plasma membrane depolarization (reviewed by Deisseroth, 2011, ibid).
Several intrinsic properties of the four known channelrhodopsins limit their application as optogenetic tools (reviewed in Lin, J. Y. A user's guide to channelrhodopsin variants: features, limitations and future developments. Exp. Physiol. 96:19-25, 2010; Hegemann, P., and A. Moglich. Channelrhodopsin engineering and exploration of new optogenetic tools. Nat. Methods. 8:39-42, 2011). The most widely used ChR2 has maximal spectral sensitivity at 470 nm but excitation at longer wavelengths is preferable to minimize light scattering by biological tissues. VChR1 is a red-shifted channelrhodopsin variant, but it has slower current kinetics compromising the fidelity of neuronal spiking at moderate to high stimulation frequencies. Another limiting property is that photocurrents generated by all channelrhodopsins in response to a pulse of continuous light decrease to a plateau level, a process called “inactivation”. In the most commonly used ChR2 this decrease can be as large as 80% from the peak amplitude, which correspondingly decreases the light-induced membrane depolarization, requiring more intense or longer light pulses to trigger neuronal action potentials or induce other biological action. This limitation is further aggravated by low unitary conductance of channelrhodopsins, which is less than that of common ion channels, as estimated by the whole-cell current noise analysis (Feldbauer, K., D. Zimmermann, V. Pintschovius, J. Spitz, C. Bamann, and E. Bamberg. Channelrhodopsin-2 is a leaky proton pump. Proc. Natl. Acad. Sci. USA. 106:12317-12322, 2009; Lin, J. Y., M. Z. Lin, P. Steinbach, and R. Y. Tsien. Characterization of engineered channelrhodopsin variants with improved properties and kinetics. Biophys. J. 96:1803-1814, 2009).